The olfactory receptors are a class of G-protein coupled receptors (GPCR) found in the membranes of cells in olfactory or other sensing organs. The olfactory receptor proteins are considered the largest sub-family of GPCR; it has been estimated that human olfactory epithelium contains approximately 2000-3000 distinct olfactory receptors, whereas insects such as Drosophila have 100-200 such receptors. It is believed that any given olfactory or sensory cell contains one or a few types of olfactory receptors.
Olfactory receptor proteins have a distinctive structure of seven hydrophobic segments that span the cell membrane (trans-membrane domains I-VII), separated by hydrophilic segments which project into the intra- or extra-cellular space. Transmembrane domains II through VII comprise a hypervariable segment which defines the ligand specificity of the receptor. This hypervariable segment is flanked by hydrophobic consensus sequences. Furthermore, it is known that the N-terminal segment of the olfactory receptor is involved in receptor stability and the C-terminal segment is involved in G-protein coupling and activation. The structural organization of a typical olfactory receptor is given in FIG. 1.
The basic olfactory signaling unit consists of an olfactory receptor, a signal transducer (e.g., G-protein), an effector (e.g., adenylyl cyclase), second messengers (e.g., cAMP), and a gated channel (e.g., a calcium channel) as depicted in FIG. 2. Olfactory receptor signaling is not, however, limited to the G-protein-adenylyl cyclase-cAMP pathway; there is evidence of olfactory receptor signaling via G-protein activation of phosphoinositidase C, with subsequent production of inositol 1,4,5-triphosphate and 1,2-diacylglycerol second messengers.
The method of chemical detection by olfactory receptors is conserved among species. Upon activation by an olfactant, an olfactory receptor initiates a cellular signaling cascade that results in the influx of calcium ions, which in turn leads to a depolarization of a connected sensory neuron. The time from receptor activation by binding of ligand to calcium influx is typically a few milliseconds.
The olfactory receptors are highly sensitive and selective; for example, they can detect femtomolar concentrations (10−13 M) of a specific chemical molecule and distinguish between two molecules differing in a single hydrogen atom.
The great variety, exquisite specificity and sensitivity, and fast-acting properties of the olfactory receptors make them ideal components of a biosensor. As its name implies, a biosensor is a detector that has a biological sensory component, such as a receptor protein or nucleic acid. Biosensors offer the advantages of higher resolution and the possibility of real-time monitoring of environments over conventional analytical techniques.
Also, because a biosensor can be constructed at the cellular or molecular level, many biosensors capable of detecting one or more substances can be contained in a very small area. Modem molecular biology and genetic techniques also allow a large number of diverse biological sensors to be generated quickly and cheaply.
However, several characteristics of typical eukaryotic expression systems, and of naturally occurring olfactory receptors, have prevented the production of a robust biological sensor that can be easily adapted to detect diverse substances. In particular, cloning and expression of olfactory receptors has been inhibited by the inability of many host cells to properly process and transport the receptors to the cell membrane.
Even if the olfactory receptors are properly positioned by the host cell, they are often not coupled to an appropriate second-messenger system. Coupling of olfactory receptors to their effectors appears to be highly specific, and endogenous G-proteins in heterologous host cells may not efficiently transduce and amplify the olfactory receptor's signal upon ligand binding. To overcome this shortcoming, host cells such as melanophores expressing large numbers of endogenous Galpha subunits (thus increasing the probability of an effective coupling) are often used. See TS McClintock et al. (1997), Molec. Brain Res. 48: 270-278. Alternatively, Galpha subunits which couple to a variety of receptors, such as the Gα15,16 subunits, are co-transfected into the host cell with the olfactory receptor. See Offermans and Simon (1995), J. Biol. Chem. 270(25): 15175-15180. However, such techniques do not reliably couple every olfactory receptor to a second-messenger.
Moreover, eukaryotic expression systems typically consist of cultured mammalian cells. Such systems are not robust, and require specialized handling under laboratory culture conditions to be viable.
The increasing threat of chemical and biological warfare agents in war and terrorist acts requires a biosensor that can operate in urban areas and battlefields alike under field conditions. The biosensor must be portable, rugged, sensitive, and reliable. What is needed, therefore, is a biosensor that can operate under the environmental conditions expected to be encountered outside the laboratory, and which reliably and reproducibly detects olfactants of interest.